Increasing toxicological and epidemiological studies have demonstrated that ambient particulate matter (PM) could cause adverse health effects including inflammation in the lung. Alveolar macrophages represent a major type of innate immune responses to foreign substances. However, the detailed mechanisms of inflammatory responses induced by PM exposure in macrophages are still unclear. We observed that coarse PM treatment rapidly activated mechanistic target of rapamycin (MTOR) in mouse alveolar macrophages in vivo, and in cultured mouse bone marrow–derived macrophages, mouse peritoneal macrophages, and RAW264.7 cells. Pharmacological inhibition or genetic knockdown of MTOR in bone marrow–derived macrophages leads to an amplified cytokine production upon PM exposure, and mice with specific knockdown of MTOR or ras homolog enriched in brain in myeloid cells exhibit significantly aggregated airway inflammation. Mechanistically, PM activated MTOR through modulation of ERK, AKT serine/threonine kinase 1, and tuberous sclerosis complex signals, whereas MTOR deficiency further enhanced the PM-induced necroptosis and activation of subsequent NF κ light-chain–enhancer of activated B cells (NFKB) signaling. Inhibition of necroptosis or NFKB pathways significantly ameliorated PM-induced inflammatory response in MTOR-deficient macrophages. The present study thus demonstrates that MTOR serves as an early adaptive signal that suppresses the PM-induced necroptosis, NFKB activation, and inflammatory response in lung macrophages, and suggests that activation of MTOR or inhibition of necroptosis in macrophages may represent novel therapeutic strategies for PM-related airway disorders.

Ambient air pollution, especially particulate matter (PM), is known to cause adverse health effects. Increasing literature from toxicological and epidemiological studies has well documented that the lung is a major target for ambient air pollutants (1, 2). Inflammation in the lungs, caused by deposited particles, can be seen as a key process that mediates adverse effects on the whole body (35). Subtypes of atmospheric particles mainly include inhalable coarse particles, which are particles with a diameter between 2.5 and 10 μm (PM10), fine particles with a diameter of 2.5 μm or less (PM2.5), and ultrafine particles (PM0.1). Researchers have focused on understanding the health effects of PM2.5 as it can penetrate deep into the alveolar regions of the lungs where gas exchange occurs, causing systemic inflammation and oxidative stress (6). However, coarse particles also very likely have health effects because they could deposit in the tracheobronchial and upper airways of the respiratory tract (68). Actually, it is reported that coarse PM may be involved in obstructive lung diseases such as asthma and chronic obstructive pulmonary disease, and emerging evidence shows that short-term coarse PM exposure may be associated with cardiovascular and respiratory morbidity (1, 9).

Pulmonary epithelial cells form the first line of human airway defense against foreign irritants, and represent the primary injury target of these pathogenic assaults (10, 11). However, accumulating studies have suggested the important roles of macrophages in dealing with outside invaders (1214). Alveolar macrophages (AMs) are phagocytes that play a critical role in homeostasis, host defense, the response to foreign substances, and tissue remodeling (15). Activity of the AMs is relatively high, because they are located at the major boundaries between the body and the outside world. We believe that AMs should play an important role in PM-induced airway inflammation, especially for the larger-sized coarse PM. However, the potential effects and detailed mechanisms of macrophages in regulation of PM-induced inflammatory responses are still unclear.

The protein kinase mechanistic target of rapamycin (MTOR) is a central regulator that controls cell growth, metabolism, and aging in response to nutrients, cellular energy stage, and growth factors (1618). MTOR links with other proteins and serves as a core component of two distinct protein complexes, MTOR complex 1 (MTORC1) and MTORC2, which regulate different cellular processes (19). Ras homolog enriched in brain (RHEB) is a GTP-binding protein that is ubiquitously expressed in humans and other mammals. RHEB localizes at the lysosome and Rag7 proteins recruit mTORC1 to the lysosome, allowing RHEB to activate the protein (20, 21). RHEB acts as an activator for mTORC1 in its GTP-bound form and is largely involved in the mTOR pathway. Emerging evidence suggests that MTORC1 activation is associated with various signaling pathways including the downstream target ribosomal protein S6 (rpS6) and inflammatory responses. However, increasing literature has recognized that the role of MTOR in disease pathogenesis is extremely cell and pathogen dependent (2224). Moreover, the functions and the underlying mechanisms by which MTOR interrelates in PM-induced inflammatory response in macrophages are not yet fully understood.

This study aims to explore the functions and the detailed mechanisms of MTOR in the regulation of PM-induced inflammatory responses in macrophages using both pharmacological and genetic approaches in vivo and in vitro. We demonstrate that MTOR serves as an early adaptive signaling that suppresses the PM-induced necroptosis, activation of NF κ light-chain–enhancer of activated B cells (NFKB), and inflammatory response in lung macrophages.

Abs against phosphorylated (p-)MTOR (5536), MTOR (2972), rpS6 (2217), p-rpS6 (4858), NF of κ light polypeptide gene enhancer in B cells inhibitor α (IκBα) (4814), p-IκBα (9246), REL-associated protein (RELA) (8242), p-RELA (4812), receptor-interacting serine/threonine-protein kinase (RIPK) 1 (3493), p–AMP-activated protein kinase α (AMPKα) (2535), p-ERK1/2 (4370), and p–tuberous sclerosis complex 2 (TSC2) (3611) were obtained from Cell Signaling Technology. NFKB p105/p50 (ab32360), RIPK3 (ab56164), and p-AKT (ab81283) were purchased from Abcam. ACTB (sc-47778) was purchased from Santa Cruz Biotechnology. All these Abs were diluted in 5% nonfat dried milk or 5% BSA (Sangon Biotech, Shanghai, China) according to manufacturer’s protocol. Anti-mouse CD45 FITC (85-11-0451-82), F4/80 Ag PE-Cyanine7 (85-25-4801-82), CD11b PerCP-Cyanine5.5 (85-45-0112-82), anti-human/mouse phospho-mTOR (S2448) PE (85-12-9718-42), and phospho-S6 ribosomal (S235/S236) APC (85-17-9007-41) used in flow cytometry were from eBioscience. All primers used in the study were synthesized by Sangon Biotech. DMEM was from GE Healthcare (SH30243; Hyclone), and FBS was purchased from Life Technologies (10270). GSK’872 (HY-101872) and IKK 16 (HY-13687) were obtained from Medchem Express, and the final concentrations in culture medium were 5 and 0.5 μM, respectively. Rapamycin (S1039), torin 1 (S2827), and necrostatin-1 (S8037) were from Selleck, and the final concentrations in the culture medium were 1.25, 125 nM, and 10 μM respectively.

PM used in the current study was purchased from the National Institute of Standards and Technology (NIST) (SRM 1649b - Urban Dust). The particle size distribution provided by the company is shown in Supplemental Fig. 1, and the estimated volume median diameter of the particle is around 2.5–10 μm according to the distribution plot. PM was suspended and sonicated for 1 h according to manufacturer’s instructions in sterile saline or sterile phosphate buffer saline to a final concentration at 2 mg/ml for in vivo or in vitro experiments.

RAW264.7 cells were purchased from American Type Culture Collection (TIB-71) and were maintained in DMEM containing 10% (v/v) FBS.

The isolation and culture of bone marrow–derived macrophages (BMDM) were performed as described previously (25) with some minor adaptions. Briefly, 6–8 wk old mice were sacrificed by cervical dislocation and soaked in 75% ethanol. The femurs and tibias were harvested and the BM cells from all bones were flushed out. After centrifuging for 5 min at 400 × g, erythrocytes were eliminated using RBC Lysing Buffer (Sigma-Aldrich). The remaining cells were seeded in plates and were incubated in DMEM containing 10% (v/v) heat-inactivated FBS and 10 ng/ml recombinant mouse M-CSF (R&D Systems) for 7 d to form proliferative nonactivated cells. Primary mouse peritoneal macrophages were obtained from the peritoneal exudates of 6–8 wk old mice, which received 1 ml fluid thioglycolate medium for three times through i.p. injection. The peritoneal exudate cells were washed twice with PBS and were cultured in DMEM for 3–4 h at 37°C and 5% CO2. The nonadherent cells were removed by washing with warm PBS.

AMs were obtained by utilizing three instillations of 0.8 ml PBS injected into the lungs. Cells were stained with CD45, CD11b, and F4/80 Abs, followed by fixation and permeabilization (BD Biosciences). Then, cells were further stained with p-MTOR or p-rpS6 Abs. CD45+CD11b+F4/80+ cells were gated to analyze the intracellular expression of p-MTOR or p-rpS6 by Cytoflex (Beckman Coulter, Brea, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR).

To establish a mouse model of acute airway inflammation in vivo, mice were treated with 100 mg PM (in 50 ml saline) per day by intratracheal instillation for 2 d. Control mice received the same volume of saline. In vitro, BMDMs, peritoneal macrophages, and RAW264.7 cells were treated with PM at various concentrations.

Wild-type C57BL/6 mice were purchased from the Animal Center of Slaccas (Shanghai, China). Mtorfl/fl and Rhebfl/fl mice on a background of C57BL/6 were purchased from the Jackson Laboratory, and LysMCre mice on a C57BL/6 background were a generous gift from Dr. G. Feng (University of California at San Diego, CA). Myeloid cell-specific MTOR and RHEB conditional knockout mice (Mtorfl/fl-LysMCre and Rhebfl/flLysMCre) were obtained by crossing Mtorfl/fl and Rhebfl/fl mice with those expressing Cre recombinase under the control of the lysozyme promoter (LysMCre). Age- and gender-matched LysMCre-negative, Mtorfl/fl and Rhebfl/fl littermates served as the control. For the in vitro BMDM experiments 4–5 wk old mice were usually used. For in vitro peritoneal macrophage experiments and in vivo PM exposure models 6–8 wk old mice were used. Primers used to identify genetically modified mice are listed in Table I. All mice were maintained in a specific pathogen-free facility. All experimental manipulations were approved by Zhejiang University Medical Laboratory Animal Care and Use Committee and Ethics Committee for Animal Studies at Zhejiang University.

After treatment with PM, BMDMs, peritoneal macrophages, and RAW264.7 cells were lysed in radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime) containing protease (04-693-116-001; Roche Diagnostics) and phosphatase inhibitors (04-906-837-001; Roche Diagnostics). Lysates were run on gels and immunoblotted with relevant Abs using standard methods. ACTB served as a protein loading control.

RNA from BMDMs and lung homogenates was isolated using RNAiso Plus (9109; Takara Bio). After isolation, the quality of the RNA was assessed with the NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific) according to the manufacturer’s instructions. The 260/280 absorbance ratios of 1.8–2.0 indicated a pure RNA sample. Reverse transcription was performed with Reverse Transcription Reagents (DRR037A; Takara Bio). The expressions of mouse Cxcl2, Il6, Tnf, and Il1β were measured by quantitative real-time PCR using SYBR Green Master Mix (DRR041A; Takara Bio) on a StepOne real-time PCR system (Applied Biosystems, Foster City, CA). A ΔΔ cycle threshold method was used to quantify the mRNA levels. All protocols were performed according to the manufacturer’s instructions. The primers used in the current study are listed in Table II.

The secreted protein levels of chemokine ligand 2 (CXCL2) (MM200; R&D Systems), IL-6 (M6000B; R&D Systems), TNF (MTA00B; R&D Systems), and IL-1β (MLB00C; R&D Systems) in cell culture supernatants or bronchoalveolar lavage fluid (BALF) supernatants were determined with ELISA kits following the manufacturer’s protocols.

Briefly, 24 h after the last exposure to PM, BALF was obtained utilizing three instillations of 0.4 ml PBS injected into the lungs and was withdrawn to collect the cells. The total number of BALF cells was counted, and then the remaining BALF was centrifuged at 3000 × g for 10 min at 4°C. The supernatants were stored at −80°C and were used for analysis of cytokines. The cell pellets were suspended in 200 μl PBS, and 40 μl of the suspension was spun onto glass microscope slides. Cells were stained with Wright–Giemsa stain (BA-4017; Baso), and differential counts were determined by counting 200 total cells.

After treatment with PM, the lungs were collected and fixed in formalin for 24 h. Then these collected lungs were embedded in paraffin and were stained with H&E following standard protocols (26, 27). Inflammation was assessed according to published guidelines.

Results are presented as mean ± SEM. Data were analyzed with GraphPad Prism 7.00 (GraphPad Software, San Diego, CA). Differences between groups were identified using one-way ANOVA. A value of p <0.05 was considered to be statistically significant.

To determine whether MTOR was biologically modulated in macrophages upon PM exposure, we first examined the expression of MTOR and related molecules in vivo and in vitro. The expressions of p-MTOR and p-rpS6 were significantly increased in AMs 1 h after in vivo PM exposure as indicated by mean fluorescence intensity (Fig. 1A–D). It is notable that the activation of MTOR was quite prompt and transient, and the levels of p-MTOR and p-rpS6 were gradually returned to basal levels at 3 or 6 h post-PM exposure. To identify whether the activation pattern of MTOR was AM specific, or if it can be applied to other macrophages, we next performed a series of in vitro experiments. Not surprisingly, the same pattern for increased p-MTOR and p-rpS6 expression was also observed in BMDMs, peritoneal macrophages, or a murine macrophage cell line RAW264.7 (Fig. 1E–G). The PM-induced expression p-MTOR in these macrophages was again transient, which was peaked at 1 h and decreased thereafter. It is worth noticing that despite the rapid and transient activation, it acted coordinately to the well-elucidated upstream signaling of MTOR activation (16). As shown in Fig. 1E, the phosphorylated levels of ERK1/2 and AKT serine/threonine kinase 1 (AKT) were increased, whereas those of AMPK were decreased upon PM exposure. These factors cooperatively resulted in the downregulation of p-TSC2 and subsequent MTOR activation. Taken together, these findings imply that PM activates MTOR signaling in macrophages.

FIGURE 1.

PM activates MTOR in macrophages both in vivo and in vitro. Mice were treated with PM for the indicated hours, and the CD45+CD11b+F4/80+ BALF cells were gated for further analysis of p-MTOR and p-rpS6. (A and C) Representative flow cytometric histograms of p-MTOR and p-rpS6 expression on alveolar macrophages from PM-treated C57BL/6 mice. (B and D) Quantified mean fluorescence intensity (MFI) of p-MTOR and p-rpS6. (E) Time-dependent expression of p-AKT, p-ERK1/2, p-AMPKα, p-TSC2, p-MTOR, MTOR, p-rpS6, and rpS6 in BMDMs stimulated with PM at 20 μg/ml. (F and G) Time-dependent expression of p-MTOR, MTOR in peritoneal macrophages, or RAW264.7 cells treated with PM at 50 μg/ml. Flow cytometric histograms are representative of five independent mice. MFI data are presented as mean ± SEM of five independent mice. Western blot data are representative of three independent experiments, and ACTB serves as loading control. *p < 0.05, ***p < 0.001. n.s., not significant (p > 0.05).

FIGURE 1.

PM activates MTOR in macrophages both in vivo and in vitro. Mice were treated with PM for the indicated hours, and the CD45+CD11b+F4/80+ BALF cells were gated for further analysis of p-MTOR and p-rpS6. (A and C) Representative flow cytometric histograms of p-MTOR and p-rpS6 expression on alveolar macrophages from PM-treated C57BL/6 mice. (B and D) Quantified mean fluorescence intensity (MFI) of p-MTOR and p-rpS6. (E) Time-dependent expression of p-AKT, p-ERK1/2, p-AMPKα, p-TSC2, p-MTOR, MTOR, p-rpS6, and rpS6 in BMDMs stimulated with PM at 20 μg/ml. (F and G) Time-dependent expression of p-MTOR, MTOR in peritoneal macrophages, or RAW264.7 cells treated with PM at 50 μg/ml. Flow cytometric histograms are representative of five independent mice. MFI data are presented as mean ± SEM of five independent mice. Western blot data are representative of three independent experiments, and ACTB serves as loading control. *p < 0.05, ***p < 0.001. n.s., not significant (p > 0.05).

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We next aimed to explore the function of MTOR in PM-induced inflammatory response in macrophages. We generated BMDMs from wild-type mice, and used two potent and widely admissive pharmacological MTOR inhibitors, rapamycin and torin 1, to avoid contingency. Interestingly, the PM-induced mRNA transcripts (Fig. 2A–D) and secreted protein levels (Fig. 2E–H) of mouse CXCL2 (C-X-C motif), IL-6, TNF-α, and IL-1β were remarkably upregulated in BDMDs treated with MTOR inhibitors, despite the fact that rapamycin is a strong immune suppressor. Primers used for quantitative PCR analysis are provided in Table II.

FIGURE 2.

Pharmacological inhibition of mTOR in BMDMs deteriorates PM-induced cytokine production. Cells were cotreated with 20 μg/ml PM and the indicated MTOR inhibitor or DMSO for 24 h. Cells were then harvested for analyzing the mRNA expression (AD) and secreted protein (EH) in cell culture supernatants of CXCL2, IL-6, TNF, IL-1B by quantitative real-time PCR and ELISA. DMSO, dimethyl sulphoxide, serves as vehicle control. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Pharmacological inhibition of mTOR in BMDMs deteriorates PM-induced cytokine production. Cells were cotreated with 20 μg/ml PM and the indicated MTOR inhibitor or DMSO for 24 h. Cells were then harvested for analyzing the mRNA expression (AD) and secreted protein (EH) in cell culture supernatants of CXCL2, IL-6, TNF, IL-1B by quantitative real-time PCR and ELISA. DMSO, dimethyl sulphoxide, serves as vehicle control. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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Table I.
Primers used for mouse identification
GenesPrimer Sequence (5′ - 3′)
Mtor Forward: 5′-TTATGTTTGATAATTGCAGTTTTGGCTAGCAGT-3′ 
 Reverse: 5′-TTTAGGACTCCTTCTGTGACATACATTTCCT-3′ 
Rheb Forward: 5′-GCCCAGAACATCTGTTCCAT-3′ 
 Reverse: 5′-GGTACCCACAACCTGACACC--3′ 
LysMCre Common: 5′-CTTGGGCTGCCAGAATTTCTC-3′ 
 WT: 5′-TTACAGTCGGCCAGGCTGAC-3′ 
 Mutant: 5′-CCCAGAAATGCCAGATTACG-3′ 
GenesPrimer Sequence (5′ - 3′)
Mtor Forward: 5′-TTATGTTTGATAATTGCAGTTTTGGCTAGCAGT-3′ 
 Reverse: 5′-TTTAGGACTCCTTCTGTGACATACATTTCCT-3′ 
Rheb Forward: 5′-GCCCAGAACATCTGTTCCAT-3′ 
 Reverse: 5′-GGTACCCACAACCTGACACC--3′ 
LysMCre Common: 5′-CTTGGGCTGCCAGAATTTCTC-3′ 
 WT: 5′-TTACAGTCGGCCAGGCTGAC-3′ 
 Mutant: 5′-CCCAGAAATGCCAGATTACG-3′ 
Table II.
Primers used for quantitative PCR analysis
GenesPrimer Sequence (5′ - 3′)
Actb Forward: 5′-GGCTGTATTCCCCTCCATCG-3′ 
 Reverse: 5′-CCAGTTGGTAACAATGCCATGT-3′ 
Cxcl2 Forward: 5′-TGTCCCTCAACGGAAGAACC-3′ 
 Reverse: 5′-CTCAGACAGCGAGGCACATC-3′ 
Il6 Forward: 5′-CTGCAAGAGACTTCCATCCAG-3′ 
 Reverse: 5′-AGTGGTATAGACAGGTCTGTTGG-3′ 
Tnf-α Forward: 5′-CTGAACTTCGGGGTGATCGG-3′ 
 Reverse: 5′-GGCTTGTCACTCGAATTTTGAGA-3′ 
Il1β Forward: 5′-CCTCCTTGCCTCTGATGG-3′ 
 Reverse: 5′-AGTGCTGCCTAATGTCCC-3′ 
GenesPrimer Sequence (5′ - 3′)
Actb Forward: 5′-GGCTGTATTCCCCTCCATCG-3′ 
 Reverse: 5′-CCAGTTGGTAACAATGCCATGT-3′ 
Cxcl2 Forward: 5′-TGTCCCTCAACGGAAGAACC-3′ 
 Reverse: 5′-CTCAGACAGCGAGGCACATC-3′ 
Il6 Forward: 5′-CTGCAAGAGACTTCCATCCAG-3′ 
 Reverse: 5′-AGTGGTATAGACAGGTCTGTTGG-3′ 
Tnf-α Forward: 5′-CTGAACTTCGGGGTGATCGG-3′ 
 Reverse: 5′-GGCTTGTCACTCGAATTTTGAGA-3′ 
Il1β Forward: 5′-CCTCCTTGCCTCTGATGG-3′ 
 Reverse: 5′-AGTGCTGCCTAATGTCCC-3′ 

To further examine the role of MTOR in regulation of inflammatory response in macrophages in vivo and in vitro, we used Mtorfl/fl-LysMCre and Rhebfl/fl-LysMCre mice, which exhibit impaired Mtor expression in myeloid cells (2830). Primers used for mouse identification are provided in Table I. We first generated BMDMs from Mtorfl/fl-LysMCre mice and their Mtorfl/fl littermates, which serve as control. As expected, Mtor-deficient BMDMs exhibited amplified cytokine production including Cxcl2, Il6, Tnf, and Il1β (Fig. 3A–D, 3I–L) compared with control. A nearly identical amplified cytokine production could also be detected in Rheb-deficient BMDMs (Fig. 3E–H, 3M–P).

FIGURE 3.

Mtor- and Rheb-deficient BMDMs exhibit amplified cytokine production upon PM treatment. BMDMs were generated from Mtorfl/fl-LysMCre (AD) or Rhebfl/fl-LysMCre (EH) mice, and were treated with PM at 20 μg/ml for 24 h. Cells were then harvested for analyzing the mRNA expression (A–H) and secreted protein (IP) in cell culture supernatants of CXCL2, IL-6, TNF, IL-1B by quantitative real-time PCR and ELISA. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Mtor- and Rheb-deficient BMDMs exhibit amplified cytokine production upon PM treatment. BMDMs were generated from Mtorfl/fl-LysMCre (AD) or Rhebfl/fl-LysMCre (EH) mice, and were treated with PM at 20 μg/ml for 24 h. Cells were then harvested for analyzing the mRNA expression (A–H) and secreted protein (IP) in cell culture supernatants of CXCL2, IL-6, TNF, IL-1B by quantitative real-time PCR and ELISA. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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Next, we investigated the role of macrophage-localized MTOR signaling in regulation of PM-induced inflammatory response in vivo. Instillation of PM at 100 μg/d per mouse for 2 d (31) increased the number of total inflammatory cells and neutrophils in BALF, both of which were further exacerbated in the Mtorfl/fl-LysMCre (Fig. 4A, 4B) or Rhebfl/fl-LysMCre mice (Fig. 5A, 5B). Inflammatory cytokines, such as CXCL2, IL-6, TNF, and IL-1β, were also notably increased in Mtorfl/fl-LysMCre (Fig. 4C–J) or Rhebfl/fl-LysMCre mice (Fig. 5C–J) in response to PM exposure. Moreover, histological analyses further confirmed that the PM-induced airway inflammation was significantly deteriorated in mice lacking MTOR (Fig. 4K, 4L) or RHEB (Fig. 5K, 5L) in myeloid cells.

FIGURE 4.

Mtorfl/fl-LysMCre mice display increased airway inflammation in response to PM exposure. Mtorfl/fl-LysMCre mice and Mtorfl/fl littermates (n = 5–8 for each group) were instilled intratracheally with PM at 100 μg/d for 2 d, and after 24 h, the total inflammatory cells (A) and the number of neutrophils (B) in the BALF were measured. Expression of the mRNA levels of Cxcl2 (C), Il6 (D), Tnf (E), and Il1β (F) in lung tissue was analyzed by quantitative PCR. Protein levels of CXCL2 (G), IL-6 (H), TNF (I), and IL-1β (J) in the BALF were measured by ELISA. (K) Representative images of lung sections stained with H&E. (L) Semiquantified inflammation score of the H&E staining (n = 10 images for each group). Data are presented as mean ± SEM of three independent experiments. Scale bar, 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Mtorfl/fl-LysMCre mice display increased airway inflammation in response to PM exposure. Mtorfl/fl-LysMCre mice and Mtorfl/fl littermates (n = 5–8 for each group) were instilled intratracheally with PM at 100 μg/d for 2 d, and after 24 h, the total inflammatory cells (A) and the number of neutrophils (B) in the BALF were measured. Expression of the mRNA levels of Cxcl2 (C), Il6 (D), Tnf (E), and Il1β (F) in lung tissue was analyzed by quantitative PCR. Protein levels of CXCL2 (G), IL-6 (H), TNF (I), and IL-1β (J) in the BALF were measured by ELISA. (K) Representative images of lung sections stained with H&E. (L) Semiquantified inflammation score of the H&E staining (n = 10 images for each group). Data are presented as mean ± SEM of three independent experiments. Scale bar, 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

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FIGURE 5.

Rhebfl/fl-LysMCre mice show aggravated airway inflammation in response to PM exposure. Rhebfl/fl-LysMCre mice and Rhebfl/fl littermates (n = 5–8 for each group) were instilled intratracheally with PM at 100 μg/d for 2 d, and after 24 h, the total inflammatory cells (A) and the number of neutrophils (B) in the BALF were measured. Expression of the mRNA levels of Cxcl2 (C), Il6 (D), Tnf (E), and Il1β (F) in lung tissue were analyzed by quantitative PCR. Protein levels of CXCL2 (G), IL-6 (H), TNF (I), and IL-1β (J) in the BALF were measured by ELISA. (K) Representative images of lung sections stained with H&E. (L) Semiquantified inflammation score of the H&E staining (n = 10 images for each group). Data are presented as mean ± SEM of three independent experiments. Scale bar, 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Rhebfl/fl-LysMCre mice show aggravated airway inflammation in response to PM exposure. Rhebfl/fl-LysMCre mice and Rhebfl/fl littermates (n = 5–8 for each group) were instilled intratracheally with PM at 100 μg/d for 2 d, and after 24 h, the total inflammatory cells (A) and the number of neutrophils (B) in the BALF were measured. Expression of the mRNA levels of Cxcl2 (C), Il6 (D), Tnf (E), and Il1β (F) in lung tissue were analyzed by quantitative PCR. Protein levels of CXCL2 (G), IL-6 (H), TNF (I), and IL-1β (J) in the BALF were measured by ELISA. (K) Representative images of lung sections stained with H&E. (L) Semiquantified inflammation score of the H&E staining (n = 10 images for each group). Data are presented as mean ± SEM of three independent experiments. Scale bar, 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

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Finally, we intended to explore the downstream pathways that mediate the role of MTOR in regulation of PM-induced inflammatory response in macrophages. IκBα is one member of a family of cellular proteins that function to inhibit the NFKB transcription factor (32). RELA, also known as p65, is a RELA involved in NFKB heterodimer formation, nuclear translocation, and activation (33, 34). Phosphorylation of RELA is a crucial posttranslational modification required for NFKB activation (35). Interestingly, expression of p-IκBα and p-RELA was significantly upregulated upon PM exposure in BMDMs, both of which were further aggravated by the knockdown of MTOR. However, the another NFKB homodimer, NFKB p105/p50, was not significantly changed either upon PM exposure or MTOR depletion (Fig. 6A).

FIGURE 6.

MTOR suppresses PM-induced NFKB activation and cytokine production through inhibition of necroptosis in BMDMs. BMDMs were generated from Mtorfl/fl and Mtorfl/fl-LysMCre mice. (A) Cells were treated with 20 μg/ml PM for the indicated time, and the expression of MTOR, RIPK1, RIPK3, IκBα, p-IκBα, RELA, p-RELA, and p105/p50 were measured by Western blot. (B) Wild-type BMDMs were treated with or without PM (20 μg/ml), and DMSO, necrostatin-1 (10 μM), and GSK’872 (5 μM) as indicated, the expression of p-IκBα and p-RELA were measured by Western blot. (CF) Mtorfl/fl and Mtorfl/fl-LysMCre mice BMDMs were treated with or without PM (20 μg/ml), DMSO, necrostatin-1 (10 μM), and GSK’872 (5 μM) and IKK 16 (0. 5 μM) as indicated. Cells were then harvested for analyzing the mRNA expression of Cxcl2, Il6, Tnf, and Il1β by quantitative real-time PCR. (GJ) Protein levels of CXCL2, IL-6, TNF, and IL-1β in the cell culture supernatants were measured by ELISA. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

MTOR suppresses PM-induced NFKB activation and cytokine production through inhibition of necroptosis in BMDMs. BMDMs were generated from Mtorfl/fl and Mtorfl/fl-LysMCre mice. (A) Cells were treated with 20 μg/ml PM for the indicated time, and the expression of MTOR, RIPK1, RIPK3, IκBα, p-IκBα, RELA, p-RELA, and p105/p50 were measured by Western blot. (B) Wild-type BMDMs were treated with or without PM (20 μg/ml), and DMSO, necrostatin-1 (10 μM), and GSK’872 (5 μM) as indicated, the expression of p-IκBα and p-RELA were measured by Western blot. (CF) Mtorfl/fl and Mtorfl/fl-LysMCre mice BMDMs were treated with or without PM (20 μg/ml), DMSO, necrostatin-1 (10 μM), and GSK’872 (5 μM) and IKK 16 (0. 5 μM) as indicated. Cells were then harvested for analyzing the mRNA expression of Cxcl2, Il6, Tnf, and Il1β by quantitative real-time PCR. (GJ) Protein levels of CXCL2, IL-6, TNF, and IL-1β in the cell culture supernatants were measured by ELISA. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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Necroptosis is a programmed form of necrosis, or inflammatory cell death. The signaling pathway responsible for carrying out necroptosis is generally understood as the accumulation of RIPK 1, which recruits RIPK3 to form the necrosome (36). In accordance with NFKB activation, both RIPK1 and RIPK3 were upregulated by PM, and were further accumulated along with the depletion of MTOR, indicating a possible role of MTOR in inhibition of PM-induced necroptosis (Fig. 6A). Next, we used two potent necroptosis inhibitors, necrostatin-1 for RIPK1 and GSK’872 for RIPK3, to block necroptosis in BMDMs in vitro. Not surprisingly, the cumulative p-IKBα and p-RELA by PM were weakened when cotreated PM with necroptosis inhibitors, indicating a positive regulation of NFKB by necroptosis (Fig. 6B). To further confirm the role of necroptosis and NFKB signaling cascade in PM-induced inflammatory response, we used IκBα inhibitor IKK 16, necrostatin-1, or GSK’872 to examine whether the amplified cytokine production due to MTOR deficiency could be reversed by blockage of necroptosis or NFKB activation. As expected, MTOR-deficient BMDMs treated with these inhibitors displayed a notable reduction of PM-induced inflammatory cytokines both in mRNA transcripts (Fig. 6C–F) and in secreted protein levels (Fig. 6G–J). Together, these results suggested that MTOR deficiency further enhanced the level of necroptosis, which mediated PM-induced NFKB activation and cytokine production in macrophages.

The major findings of this study can be summarized as follows: 1) PM transiently increases the expression of MTOR and its activity through upstream ERK/AKT/AMPK/TSC signaling in macrophages; 2) activation of MTOR serves as an early adaptive signaling and suppresses the PM-induced inflammatory response; 3) mice with specific knockdown of MTOR or RHEB in myeloid cells exhibit significantly aggerated airway inflammation; and 4) the mechanism of MTOR in suppression of PM-induced inflammatory response in macrophages was likely via inhibition of necroptosis and subsequent NFKB pathways.

The adverse health effects of inhaling PM have been widely studied in respiratory diseases, cardiovascular disease, and premature death (3739), and the adverse respiratory health effects have drawn considerable worldwide attention over recent decades (5, 4043). Because particle sizes decide where the particle lands in the airway, much of the enthusiasm and effort has been drawn to PM2.5 or even PM0.1 (6). However, Keet et al. (9) have recently reported positive associations of pediatric asthma with coarse PM that persist after statistical adjustment for PM2.5. Such studies suggested that larger-sized coarse PM could not be ignored in terms of the public health burden of the airborne particles (1). The PM used in our current study, NIST-1649b, is supposed to have a mean diameter that fits to the criterion of coarse PM (2.5–10 μm) according to the certificated of analysis provided by the manufacturer. Thus, our study provides a strong evidence that coarse PM may trigger the same adverse health effects as fine or ultrafine particles.

There is no doubt that the lung is a major target for ambient air pollutants, and much of the enthusiasm in PM has been drawn to the epithelial cells, which are the first line of defense against outside irritants. However, inflammation is a protective response involving many different types of cells and molecular mediators. In the lung, resident AMs provide sentinel function against inhaled pathogens (44). Bacterial constituents, pathogens, or irritants ligate toll-like receptors on AMs (45), causing AMs to secrete proinflammatory cytokines (46) that activate alveolar epithelial receptors (47) and lead to the recruitment of neutrophils (48, 49). Therefore, due attention should be paid to macrophages (50). Our current study focused on macrophages in regulation of PM-induced pulmonary inflammation, and clearly demonstrated that modulation of certain proteins in macrophages orchestrated the overall airway inflammation in vivo. Thus, responses from AMs and the airway epithelium should cooperatively regulate the PM-induced inflammation in the lung. The further exploration of inflammatory responses in macrophages in situ and the dynamic interaction between AMs and airway epithelial cells may be of great significance in understanding the pathogenesis of PM-related pulmonary disorders.

It has been shown previously that MTOR participates in the regulation of the inflammatory response in macrophages. However, whether MTOR is up- or downregulated upon PM exposure remains inconsistent. A recent publication reported that PM exposure induced autophagy in macrophages via the oxidative stress–mediated PI3K/AKT/mTOR pathway, and showed that MTOR was downregulated upon PM exposure (23). In contrast, another study demonstrated that activation of MTOR exerted direct effects on macrophage polarizations upon airborne PM2.5 exposure (24). Our current study is inconsistent with the latter, demonstrating that PM activates MTOR in a quite early stage (Fig. 1). The discrepancy of PM-induced MTOR may lie in the sources of the PM, as researchers are more likely to use collected urban particles when conducting the experiments. In our study, PM was purchased from NIST, although it is always difficult to define standard PM. Because the elements constituting the PM do make a significant difference to the triggering of multifarious mechanisms, a more explicit criterion of PM for research may be of urgent need.

The activation and function of MTOR might be cell type–dependent. We have demonstrated in our previous studies that certain pathways, like elevated autophagy, are essential for ultrafine particle–induced inflammation and mucus hyperproduction in the airway epithelium (31), and autophagy inhibitors suppress environmental PM-induced airway inflammation (51). We have also observed a decreased MTOR level in PM-treated human bronchial epithelial cells or in lung tissue homogenate of acute PM exposure. Not surprisingly, both genetic knockdown and pharmacological inhibition of MTOR in epithelial cells led to an amplified cytokine production, indicating a protective role of MTOR in PM-induced airway inflammation (Z. Li and Yinfang Wu, unpublished observations). Despite the fact that MTOR is upregulated in macrophages in the current study, it is of interest that inhibition of MTOR in macrophages resulted in an anabatic inflammatory response and worse airway inflammation, as it did in airway epithelial cells. However, whether the autophagy level declined as a result of the activation of MTOR, or whether it serves as a downstream signal and mediates the inflammatory response in macrophages upon PM exposure remain to be further elucidated.

Necroptosis has been implicated in the pathology of many types of acute tissue damage, including myocardial infarction, stroke, and ischemia-reperfusion injury (52). Recently, mitophagy-dependent necroptosis has been shown to mediate cigarette smoke–induced airway injury (53), and cigarette smoke could also induce necroptosis and damage-associated molecular pattern release to trigger neutrophilic airway inflammation (54). Our current data demonstrated that PM exposure increased the expression of RIPK1 and RIPK3 in macrophages, suggesting an increased necroptosis by PM. It has been reported that in schwannoma cells, both AKT and MTOR can mediate necroptosis (55). In line with this, we observed that the levels of RIPK1 and RIPK3 were further enhanced along with the depletion of MTOR, indicating a possible role of MTOR in the suppression of PM-induced necroptosis. Further investigations on the detailed mechanisms of PM-induced necroptosis might be warranted.

Collectively, to our knowledge, the present study shows for the first time that activation of MTOR suppresses the PM-induced macrophage inflammatory responses in vitro and acute airway inflammation pathogenesis, likely through modulation of necroptosis and subsequent NFKB. Thus, activation of MTOR or inhibition of necroptosis in macrophages may represent novel therapeutic strategies for PM-related lung inflammation.

We thank Prof. Gensheng Feng (University of California at San Diego, CA) for providing the LysMCre mice.

This work was supported by the Key Project of Chinese National Programs for Fundamental Research and Development (973 Program, Grant 2015CB553405 to Z.C.), National Key R&D Program of China (Grant 2016YFA0501602 to Z.C.), and the Major and General Projects from the National Natural Science Foundation of China (Grants 81490532 to H.S. and 81370142 and 81670031 to Z.C.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AM

alveolar macrophage

AMPK

AMP-activated protein kinase

BALF

bronchoalveolar lavage fluid

BMDM

bone marrow–derived macrophage

CXCL2

chemokine ligand 2

IκBα

NF of κ light polypeptide gene enhancer in B cells inhibitor α

MTOR

mechanistic target of rapamycin

MTORC1

MTOR complex 1

NFKB

NF κ light-chain–enhancer of activated B cells

NIST

National Institute of Standards and Technology

p-

phosphorylated

PM

particulate matter

RELA

REL-associated protein

RHEB

ras homolog enriched in brain

RIPK

receptor-interacting serine/threonine-protein kinase

rpS6

ribosomal protein S6

TSC2

tuberous sclerosis complex 2.

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The authors have no financial conflicts of interest.

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